Multiplexed single molecule rna visualization with a two-probe proximity ligation system
AssigneeThe Board of Trustee of the Leland Stanford Junior University
InventorsNikolay Samusik, Felice Alessio Bava, Yury Goltsev, Garry P. Nolan
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Abstract
SNAIL provides cost-efficient detection of specific nucleic acids in single cells, and may be combined with flow cytometry to simultaneously analyze large numbers of cells for a plurality of nucleic acids, e.g. at least one, to up to 5, up to 10, up to 15, up to 20 or more transcripts can be simultaneously analyzed, at a rate of up to about 50, 100, 250, 500 or more cells/second. The methods require only two primers for amplification, and may further include a detection primer.
Claims23
A method for determining the abundance of a target nucleic acid in a single cell, the method comprising:
- contacting a fixed and permeabilized cell with at least one pair of SNAIL oligonucleotide primers under conditions permissive for specific hybridization, wherein the pair of primers comprises a Splint Primer Oligonucleotide (SPO) and a Padlock Oligonucleotide (PO), wherein each of SPO and PO comprise a first complementarity region (CR1 and CR1′, respectively) complementary to adjacent sequences on the target nucleic acid;
- and each of SPO and PO further comprise a second complementarity region (CR2 and CR2′) located adjacent to CR1 or CR1′;
- wherein CR2′ is a split region such that the 5′ and the 3′ ends of PO hybridize to CR2 such that after hybridization the 5′ and the 3′ ends of PO are positioned directly adjacent to one another;
- washing the cell free of unbound primer;
- contacting the cell with ligase wherein the PO is ligated to generate a closed circle;
- performing rolling circle amplification using the PO as a template and SPO as a primer for a polymerase;
- contacting the cell with a detection probe under conditions permissive for specific hybridization;
- and
- detecting the level of bound detection probes to determine the abundance of the target nucleic acid.
Dependencies are inferred from the claim text.